There is evidence that reactive nitrogen species (RNS) derived from nitric oxide (NO) of endothelial nitric oxide synthase (eNOS), such as peroxynitrite (ONOO-), are important in cardiovascular diseases including diabetes. These oxidants would not be sensitive to "classical" lipid-soluble antioxidants such as vitamin E, which might explain why antioxidant therapy is ineffective in delivering sustained improvement. However, the mechanisms by which diabetes increases RNS, and those by which RNS modifies vascular functions are poorly understood. Our preliminary studies have established new insights into how hyperglycemia and free fatty acids (FFA) increase RNS and the mechanisms by which its effects on cell function are mediated. Exposure of cultured human aortic endothelial cells (HAEC) to clinically relevant concentrations of glucose (20 mM) and FFA (up to 0.5 mM) for 3 days additively increases the production of both NO and O2-, and, consequently, decreases the bioactivity of NO, as indicated by decreased levels of cyclic GMP. Further evidence that NO is inactivated by reacting with 02- to form the reaction products, ONOO-, is found in the increased levels of its reaction product with tyrosine, 3-nitrotyrosine (3-NT). While the function of many proteins may be affected, we have found that prostacyclin synthase (PGIS) is particularly susceptible to tyrosine nitration; the levels of nitrated PGIS increases and its activity decreases in HAEC grown in hyperglycemia/FFA. This may not only explain why diabetes decreases levels of PGI2, but also why an increase has been noted in its precursor PGH2 which activates upon thromboxane A2 receptor (termed TP receptor, TPr). Our preliminary studies have also shown that activation of TPr can modulate both adhesion molecule expression and apoptosis in HAEC. In addition, either TPr antagonist or inhibition of cyclooxygenase (COX) significantly attenuates both the expression of these adhesion molecules and apoptosis, suggesting activation of TPr by PGH2 occurs in HAEC exposed to hyperglycemia/FFA. Thus, our central hypothesis is that diabetes via hyperglycemia/hyperlipdemia increases the generation of 02- and then ONOO', resulting in eNOS uncoupling, PGIS nitration, and TPr stimulation. This contributes to the initiation and progression of vascular complications in diabetes mellitus because of the down-regulation of protective actions of NO and PGI2 and because the non-metabolized PGH2 tips the balance towards platelet aggregation, atheroma accumulation, and thrombus formation. Thus, the aims of the proposed studies are: (1) To elucidate the mechanism by which hyperglycemia and FFA increases the production of NO and O2-, as well as its reaction product ONOO-, and nitration and inactivation of PGIS in cultured HAEC; (2) To determine the role of TPr stimulation in enhancing endothelial cell adhesion molecules expression and apoptosis under conditions of increased oxidant stress caused by hyperglycemia and FFA; (3) To determine if oxidant stress and PGIS inactivation contribute to the diabetes-enhanced atherogenesis in transgenic and knockout mice.